EP3703735A1 - Cellules nk-92 pour stimuler un vaccin anticancéreux - Google Patents

Cellules nk-92 pour stimuler un vaccin anticancéreux

Info

Publication number
EP3703735A1
EP3703735A1 EP18815377.9A EP18815377A EP3703735A1 EP 3703735 A1 EP3703735 A1 EP 3703735A1 EP 18815377 A EP18815377 A EP 18815377A EP 3703735 A1 EP3703735 A1 EP 3703735A1
Authority
EP
European Patent Office
Prior art keywords
tumor
cells
car
subject
expressing
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP18815377.9A
Other languages
German (de)
English (en)
Inventor
Hans G. KLINGEMANN
Laurent H. BOISSEL
Nathan SCHOMER
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Immunitybio Inc
Original Assignee
NantKwest Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by NantKwest Inc filed Critical NantKwest Inc
Publication of EP3703735A1 publication Critical patent/EP3703735A1/fr
Pending legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4631—Chimeric Antigen Receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
    • A61K38/1774—Immunoglobulin superfamily (e.g. CD2, CD4, CD8, ICAM molecules, B7 molecules, Fc-receptors, MHC-molecules)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • A61K38/2013—IL-2
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    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4613—Natural-killer cells [NK or NK-T]
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K39/4643—Vertebrate antigens
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    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/6425—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent the peptide or protein in the drug conjugate being a receptor, e.g. CD4, a cell surface antigen, i.e. not a peptide ligand targeting the antigen, or a cell surface determinant, i.e. a part of the surface of a cell
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    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70517—CD8
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/80—Vaccine for a specifically defined cancer
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/31—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterized by the route of administration
    • A—HUMAN NECESSITIES
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    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
    • A61K2239/48—Blood cells, e.g. leukemia or lymphoma
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications
    • C12N2320/32—Special delivery means, e.g. tissue-specific
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/2302—Interleukin-2 (IL-2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
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    • C12N2501/23—Interleukins [IL]
    • C12N2501/2312—Interleukin-12 (IL-12)

Definitions

  • Cancer is a leading cause of illness and death worldwide. For example, over
  • Chemotherapy involves the disruption of cell replication or cell metabolism, and it remains one of the main treatment options for cancer. Chemotherapy can be effective, but there are severe side effects, e.g., vomiting, low white blood cells (WBC), loss of hair, loss of weight and other toxic effects. Because of the extremely toxic side effects, many cancer individuals cannot successfully finish a complete chemotherapy regime. Cancer drug monotherapy also selects for mutant cancer cells that are resistant to the drug.
  • WBC white blood cells
  • Radiation therapy uses high-energy radiation to damage tumor cells' DNA, causing them to stop proliferating and/or die.
  • radiation is non-specific and kills healthy cells along with the cancerous ones.
  • Targeted radiation e.g., external beam radiation, brachytherapy
  • systemic radiation has a greater potential of harming a large number of normal cells and tissues.
  • Radiation also has negative side effects, including a risk of a secondary cancer caused by the radiation.
  • NK cells Natural killer cells are cytotoxic lymphocytes that constitute a major component of the innate immune system. NK cells, generally representing about 10-15% of circulating lymphocytes, bind and kill targeted cells, including virus-infected cells and many malignant cells, non-specifically with regard to antigen and without prior immune sensitization.
  • NK cells have been shown to be somewhat effective in both ex vivo therapy and in vivo treatment.
  • NK cells used for this purpose are isolated from the peripheral blood lymphocyte ("PBL") fraction of blood from the subject, expanded in cell culture in order to obtain sufficient numbers of cells, and then re-infused into the subject.
  • PBL peripheral blood lymphocyte
  • kits for inducing and maintaining an immune response to a tumor in a subject while treating a primary tumor include administering to the subject an effective amount of CAR-expressing-NK-92 cells to treat the primary tumor thereby inducing an anti-tumor immune response that is maintained in the subject, the maintained immune response preventing tumor regrowth and/or inhibiting generation of secondary tumors.
  • methods of producing an anti-tumor vaccine in a subject with a tumor include administering to the subject an effective amount of CAR-expressing-NK-92 cells to the subject thereby inducing an antitumor vaccine to the tumor in the subject.
  • described herein is a method for inducing and maintaining an immune response to a tumor in a subject while treating a primary tumor.
  • the method comprises administering to the subject an effective amount of CAR-expressing- K-92 cells to treat the primary tumor thereby inducing an anti-tumor immune response that is maintained in the subject, the maintained immune response preventing tumor regrowth and/or inhibiting generation of secondary tumors.
  • the method results in interleukin 6 expression being increased in the subject.
  • the CAR-expressing-NK-92 cells induce lysis of tumor cells in the primary tumor.
  • a cytokine is co-administered to the subject.
  • the cytokine is interleukin 2.
  • the cytokine is interleukin 12.
  • a chemotherapeutic agent is administered to the subject.
  • the chemotherapeutic agent is administered to the subject prior to administration of the CAR-expressing-NK-92 cells.
  • the CAR-expressing-NK-92 cells are administered to the subject prior to administration of the CAR-expressing-NK-92 cells.
  • chemotherapeutic agent is administered to the subject after administration of the CAR- expressing-NK-92 cells. In one embodiment, the chemotherapeutic agent is administered to the subject substantially simultaneously with administration of the CAR-expressing-NK-92 cells.
  • the CAR-expressing-NK-92 cells are administered systemically. In some embodiments, the CAR-expressing-NK-92 cells are administered proximate to or directly into the primary tumor.
  • the tumor is selected from the group consisting of colorectal tumor, breast tumor, lung tumor, prostate tumor, pancreatic tumor, bladder tumor, cervical tumor, cholangiocarcinoma, gastric sarcoma, glioma, leukemia, lymphoma, melanoma, multiple myeloma, osteosarcoma, ovarian tumor, stomach tumor, brain tumor.
  • the tumor is a B-cell lymphoma.
  • the method further comprises administering to the subject a cancer drug or radiation.
  • the subject is selected from the group consisting of bovines, swine, rabbits, alpacas, horses, canines, felines, ferrets, rats, mice, fowl and buffalo. In one embodiment, the subject is a human.
  • the CAR-expressing-NK-92 cells express a CD 19-
  • the NK-92 cell is modified to express a chimeric antigen receptor (CAR) on the cell surface.
  • the CAR comprises an antigen binding domain (e.g., ScFv) that specifically binds an antigen expressed by tumor cells.
  • the antigen binding domain specifically binds the CD 19 antigen.
  • the tumor cells comprise lymphoma cells.
  • the NK-92 cells express a CAR that specifically binds CD 19 and the tumor cells comprise lymphoma cells.
  • the NK-92 cells express murine CD19CAR
  • the mCD19CAR comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 3.
  • the NK- 92 cells express a codon optimized CAR on the cell surface, where the CAR is codon optimized for expression in humans.
  • the NK-92 cells express a codon optimized CD19CAR on the cell surface.
  • the codon optimized CAR is codon optimized for expression in humans.
  • CD19CAR comprises an amino acid sequence at least 90% identical to SEQ ID NO: 5.
  • the NK-92 cells express a codon optimized CD20CAR on the cell surface.
  • the codon optimized CD20CAR comprises an amino acid sequence at least 90% identical to SEQ ID NO: 7.
  • the NK-92 cells express a codon optimized CD33CAR on the cell surface.
  • the codon optimized CD33CAR comprises an amino acid sequence at least 90% identical to SEQ ID NO: 7.
  • the NK-92 cells express a codon optimized CSPG4-CAR on the cell surface. In one embodiment, the codon optimized CSPG4-CAR comprises an amino acid sequence at least 90% identical to SEQ ID NO: 11. In one embodiment, the NK-92 cells express a codon optimized EGFR-CAR on the cell surface. In one embodiment, the codon optimized EGFR-CAR comprises an amino acid sequence at least 90% identical to SEQ ID NO: 13. In one embodiment, the NK-92 cells express a codon optimized IGFIR-CAR on the cell surface. In one embodiment, the codon optimized IGFIR-CAR comprises an amino acid sequence at least 90% identical to SEQ ID NO: 15.
  • the NK-92 cells express a codon optimized CD30-CAR on the cell surface. In one embodiment, the codon optimized CD30-CAR comprises an amino acid sequence at least 90% identical to SEQ ID NO: 17. In one embodiment, the NK-92 cells express a codon optimized HER2/neu-CAR on the cell surface. In one embodiment, the codon optimized HER2/neu-CAR comprises an amino acid sequence at least 90% identical to SEQ ID NO: 19. In one embodiment, the NK- 92 cells express a codon optimized GD2-CAR on the cell surface. In one embodiment, the codon optimized GD2-CAR comprises an amino acid sequence at least 90% identical to SEQ ID NO: 22 or SEQ ID NO:23.
  • a method of producing an anti-tumor vaccine in a subject with a tumor comprising administering to the subject an effective amount of CAR-expressing-NK-92 cells thereby inducing an anti-tumor vaccine to the tumor in the subject.
  • the method results in increased expression of interleukin 6 in the subject.
  • the CAR-expressing-NK-92 cells treats the tumor in the subject.
  • a cytokine is co-administered to the subject.
  • the cytokine is interleukin 2.
  • the cytokine is interleukin 12.
  • a chemotherapeutic agent is administered to the subject.
  • the chemotherapeutic agent is administered to the subject prior to administration of the CAR-expressing-NK-92 cells.
  • the chemotherapeutic agent is administered to the subject after administration of the CAR- expressing-NK-92 cells.
  • the chemotherapeutic agent is administered to the subject substantially simultaneously with administration of the CAR-expressing-NK-92 cells.
  • the CAR-expressing-NK-92 cells are administered systemically. In some embodiments, the CAR-expressing-NK-92 cells are administered proximate to or directly into the primary tumor.
  • the tumor is selected from the group consisting of colorectal tumor, breast tumor, lung tumor, prostate tumor, pancreatic tumor, bladder tumor, cervical tumor, cholangiocarcinoma, gastric sarcoma, glioma, leukemia, lymphoma, melanoma, multiple myeloma, osteosarcoma, ovarian tumor, stomach tumor, brain tumor.
  • the tumor is a B-cell lymphoma.
  • the method further comprises administering to the subject a cancer drug or radiation.
  • the subject is selected from the group consisting of bovines, swine, rabbits, alpacas, horses, canines, felines, ferrets, rats, mice, fowl and buffalo. In one embodiment, the subject is a human.
  • the CAR-expressing-NK-92 cells are mCD19CAR- expressing NK-92 cells.
  • a CAR-expressing-NK-92 cell for use in treating a primary or secondary tumor in a subject.
  • a CAR-expressing-NK-92 cell for use in inducing and maintaining an immune response to a tumor in a subject while treating a primary tumor.
  • the use comprises administering to the subject an effective amount of CAR-expressing-NK-92 cells to treat the primary tumor thereby inducing an anti-tumor immune response that is maintained in the subject, the maintained immune response preventing tumor regrowth and/or inhibiting generation of secondary tumors.
  • Fig. 1 is a graph showing that wild type NK-92 cells produce IL-8, IL-10, and interferon gamma ( ⁇ FNy), but not assayable amounts of IL-6 as determined by qualitative ELISA assay.
  • Fig. 2A shows surface expression of mCD19CAR in cells in flow cytometry experiments.
  • Fig. 2B shows killing of murine A20 lymphoma cells in vitro by mCD19CAR- expressing NK-92 cells.
  • Fig. 3 is a graph showing reduced tumor surface area (mm 2 ) after NK-92-
  • CD19CAR administration (circles) and continued regression until the tumor is no longer visible.
  • Tumor surface area initially reduces after injection of wild type NK-92 cells (stars), but subsequently increases and tumor regrows.
  • Figs. 4A-4D show intra-tumor treatment promotes clearance of A20 tumor tumors and increases survival.
  • Fig. 4A is a schematic showing the experimental
  • Fig. 4B is a stacked bar graph depicting the percentage of tumor seeding observed in each condition. No statistically significant differences were found when a two- tailed Fisher's exact test was used to compare efficiency of tumor seeding between females and males.
  • Fig. 4C shows the change in tumor volume over time in separate graphs for each of the treatments: vehicle, parental NK-92 cells, or mCD19CAR-NK-19 cells. Each male and female for each treated group are plotted separately.
  • Fig. 5 is a bar graph showing average tumor volumes for males, females, or both on Day 16 post-treatment.
  • Fig. 6 is a graph showing survival of mice after re-challenge with A20 tumor cells. All tumor-free mice surviving by Day 30 were re-challenged by subcutaneous injection of A20 cells in the contralateral flank. All mice remained tumor-free and survived until day 60 post-treatment, except one.
  • Fig. 7 shows a Kaplan-Meier survival curve of mice injected with A20 tumor cells following intratumor treatment with mCD19-CAR K-92 cells vs. vehicle control, as described in the Examples.
  • Fig. 8 shows tumor size of complete responders vs. naive controls re- challenged with A20 tumor cells, as described in the Examples.
  • Fig. 9 shows a Kaplan-Meier survival curve of mice injected with L1210-Luc tumor cells following intratumor treatment with mCD19-CAR NK-92 cells vs. vehicle control, as described in the Examples.
  • Fig. 10 shows tumor size of complete responders vs. naive controls re- challenged with L1210-Luc tumor cells, as described in the Examples.
  • a range includes each individual member.
  • a group having 1-3 cells refers to groups having 1, 2, or 3 cells.
  • a group having 1-5 cells refers to groups having 1, 2, 3, 4, or 5 cells, and so forth.
  • compositions and methods include the recited elements, but not excluding others.
  • Consisting essentially of when used to define compositions and methods shall mean excluding other elements of any essential significance to the combination. For example, a composition consisting essentially of the elements as defined herein would not exclude other elements that do not materially affect the basic and novel characteristic(s) of the claimed subject matter.
  • Consisting of shall mean excluding more than trace amount of other ingredients and substantial method steps. Embodiments defined by each of these transition terms are within the scope of this disclosure.
  • concurrent refers to the administration of at least two agents (e.g. K-92-Fc-CAR cells and a cancer drug) at the same time or at approximately the same time
  • cancer drugs refers to chemical and biological agents used to treat cancer. Such cancer drugs include, but are not limited to, chemotherapeutic agents, hormonal therapy agents, and the like as well as combinations thereof.
  • the patient, subject, or individual is a mammal. In a particularly preferred embodiment, the patient, subject or individual is a human.
  • treating covers the treatment of a disease or disorder described herein, in a subject, such as a human, and includes: (i) inhibiting a disease or disorder, i.e., arresting its development; (ii) relieving a disease or disorder, i.e., causing regression of the disorder; (iii) slowing progression of the disorder; and/or (iv) inhibiting, relieving, or slowing progression of one or more symptoms of the disease or disorder.
  • administering or “administration” of a monoclonal antibody or a natural killer cell to a subject includes any route of introducing or delivering the antibody or cells to perform the intended function. Administration can be carried out by any route suitable for the delivery of the cells or monoclonal antibody.
  • delivery routes can include intravenous,
  • a monoclonal antibody and/or K-92 cells are administered directly to the tumor, e.g., by injection into the tumor. Administration includes self-administration and the administration by another.
  • the term "effective dose” or “effective amount” refers to a dose of an agent or composition (e.g., NK-92 cells) containing the agent that produces the desired effect(s) (e.g., treating or preventing a disease).
  • an agent or composition e.g., NK-92 cells
  • the exact dose and formulation will depend on the purpose of the treatment and will be ascertainable by one skilled in the art using known techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Remington (2012); and Pickar, Dosage Calculations (9th edition) (1999)).
  • a therapeutically effective amount will show an increase or decrease of at least 5%, 10%, 15%, 20%, 25%, 40%, 50%, 60%, 75%, 80%, 90%, or at least 100%.
  • Therapeutic efficacy can also be expressed as "-fold" increase or decrease.
  • a therapeutically effective amount can have at least a 1.2-fold, 1.5-fold, 2-fold, 5-fold, or more effect over a standard control.
  • a therapeutically effective dose or amount may ameliorate one or more symptoms of a disease.
  • a therapeutically effective dose or amount may prevent or delay the onset of a disease or one or more symptoms of a disease when the effect for which it is being administered is to treat a person who is at risk of developing the disease.
  • sequential administration refers to administration of at least two active ingredients at different times, the administration route being identical or different. More particularly, sequential use refers to the whole administration of one of the active ingredients before administration of the other or others commences. It is thus possible to administer one of the active ingredients over several seconds, minutes, hours, or days before administering the other active ingredient or ingredients.
  • therapeutic use refers to the administration of at least two active ingredients by the same or different route and at the same time or at substantially the same time.
  • the term "primary tumor” generally refers to the original tumor. Cells from the primary tumor may break off and form secondary. As a practical matter, the primary tumor is a known tumor that is desired to be treated by the cancer drugs and/or K-92 cell therapy. In a preferred embodiment, the primary tumor is located at the site of origin for the cancer. For example, a primary tumor for a breast cancer is located in the breast.
  • secondary tumor refers to a tumor that is related to (e.g., arose/metastasized from) the primary tumor but located at a site distinct from the primary tumor.
  • a secondary tumor for breast cancer may be located in the bone. Secondary tumor formation is a problem for cancer treatment.
  • NK cells are cells of the immune system that kill target cells in the absence of a specific antigenic stimulus, and without restriction according to major histocompatibility complex (MHC) class.
  • Target cells may be cancer or tumor cells.
  • NK cells are characterized by the presence of CD56 and the absence of CD3 surface markers.
  • Endogenous NK cells is used to refer to NK cells derived from a donor (or the patient), as distinguished from the NK-92 cell line. Endogenous NK cells are generally heterogeneous populations of cells within which NK cells have been enriched. Endogenous NK cells may be intended for autologous or allogeneic treatment of a patient.
  • NK-92 refers to natural killer cells derived from the highly potent unique cell line described in Gong et al. (1994), rights to which are owned by NantKwest (hereafter, "NK-92TM cells”). The immortal NK cell line was originally obtained from a patient having non-Hodgkin's lymphoma.
  • NK-92TM is intended to refer to the original NK-92 cell lines as well as NK-92 cell lines that have been modified (e.g., by introduction of exogenous genes).
  • NK-92TM cells and exemplary and non- limiting modifications thereof are described in U.S. Patent Nos. 7,618,817; 8,034,332;
  • NK-92TM cells are known to persons of ordinary skill in the art, to whom such cells are readily available from NantKwest, Inc.
  • aNK refers to an unmodified natural killer cells derived from the highly potent unique cell line described in Gong et al. (1994), rights to which are owned by NantKwest (hereafter, “aNKTM cells”).
  • haNK refers to natural killer cells derived from the highly potent unique cell line described in Gong et al. (1994), rights to which are owned by NantKwest, modified to express CD 16 on the cell surface (hereafter, "CD 16+ NK- 92TM cells” or “haNK® cells”).
  • the CD 16+ NK-92TM cells comprise a high affinity CD 16 receptor on the cell surface.
  • the high affinity CD 16 molecule contains a phenylalanine to valine substitution at codon/position 158 (F158V) of the mature CD16 peptide, which binds with higher affinity to human IgGl than does CD 16 with phenylalanine (F) at codon 158.
  • F158V codon/position 158
  • taNK refers to natural killer cells derived from the highly potent unique cell line described in Gong et al. (1994), rights to which are owned by
  • t-haNK refers to natural killer cells derived from the highly potent unique cell line described in Gong et al. (1994), rights to which are owned by NantkWest, modified to express CD 16 on the cell surface and to express a chimeric antigen receptor (hereafter, "CAR-modified CD 16+ NK-92TM cells” or “t-haNKTM cells”).
  • the t-haNKTM cells express a high affinity CD 16 receptor on the cell surface.
  • NK-92 cells have high cytotoxicity even at a low effectontarget (E:T) ratio, e.g., 1 : 1. (Gong, et al., supra).
  • E:T effectontarget
  • a "modified NK-92 cell” refers to an NK-92 cell that expresses an exogenous gene or protein, such as an Fc receptor, a CAR, a cytokine (such as IL-2 or IL-12), and/or a suicide gene.
  • the modified NK-92 cell comprises a vector that encodes for a transgene, such as an Fc receptor, a CAR, a cytokine (such as IL-2 or IL-12), and/or a suicide gene.
  • the modified NK-92 cell expresses at least one transgenic protein.
  • non-irradiated NK-92 cells are NK-92 cells that have not been irradiated. Irradiation renders the cells incapable of growth and proliferation. It is envisioned that the NK-92 cells will be irradiated at the treatment facility or some other point prior to treatment of a patient, since the time between irradiation and infusion should be no longer than four hours in order to preserve optimal activity. Alternatively, NK-92 cells may be inactivated by another mechanism.
  • inactivation of the NK-92 cells renders them incapable of growth. Inactivation may also relate to the death of the NK-92 cells. It is envisioned that the NK-92 cells may be inactivated after they have effectively purged an ex vivo sample of cells related to a pathology in a therapeutic application, or after they have resided within the body of a mammal a sufficient period of time to effectively kill many or all target cells residing within the body. Inactivation may be induced, by way of non-limiting example, by administering an inactivating agent to which the NK-92 cells are sensitive.
  • cytotoxic when used to describe the activity of effector cells such as NK cells, are intended to be synonymous.
  • cytotoxic activity relates to killing of target cells by any of a variety of biological, biochemical, or biophysical mechanisms. Cytolysis refers more specifically to activity in which the effector lyses the plasma membrane of the target cell, thereby destroying its physical integrity. This results in the killing of the target cell. Without wishing to be bound by theory, it is believed that the cytotoxic effect of K cells is due to cytolysis.
  • the term "kill" with respect to a cell/cell population is directed to include any type of manipulation that will lead to the death of that cell/cell population.
  • Fc receptor refers to a protein found on the surface of certain cells
  • Fc region e.g., natural killer cells
  • FcR Fc receptor
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • FcRs are classified based on the type of antibody they recognize. For example, Fc- gamma receptors (FcyR) bind to the IgG class of antibodies.
  • FcyRIII-A also called CD 16 is a low affinity Fc receptor bind to IgG antibodies and activate ADCC.
  • FcyRIII-A are typically found on NK cells. NK-92 cells do not express FcyRIII-A.
  • a representative polynucleotide sequence encoding a native form of CD16 is shown in SEQ ID NO: 1.
  • the high affinity Fc Receptor III-A amino acid sequence (full length) is shown in SEQ ID NO:24.
  • chimeric antigen receptor refers to an extracellular antigen-binding domain that is fused to an intracellular signaling domain.
  • CARs can be expressed in T cells or NK cells to increase cytotoxicity.
  • the extracellular antigen- binding domain is a scFv that is specific for an antigen found on a cell of interest.
  • a CAR- expressing NK-92 cell is targeted to cells expressing certain antigens on the cell surface, based on the specificity of the scFv domain.
  • the scFv domain can be engineered to recognize any antigen, including tumor-specific antigens.
  • CARs and/or scFv domains include those that recognize the following antigens: CD19 (SEQ ID NO:3, SEQ ID NO:5), CD 20 (SEQ ID NO:7); CD33 (SEQ ID NO:9), CSPG4 (SEQ ID NO: 11), EGFR (SEQ ID NO: 13), IGF1R (SEQ ID NO: 15), CD30 (SEQ ID NO: 17), HER2/neu (SEQ ID NO: 19), and GD2 (SEQ ID NO:22 (VL/VH format) or SEQ ID NO:23 (VH/VL format)).
  • polynucleotide polynucleotide
  • Polynucleotides can have any three-dimensional structure and may perform any function, known or unknown.
  • a gene or gene fragment for example, a probe, primer, EST or SAGE tag
  • exons introns
  • messenger RNA messenger RNA
  • transfer RNA transfer RNA
  • ribosomal RNA ribozymes
  • cDNA recombinant polynucleotides
  • branched polynucleotides plasmids
  • a polynucleotide can comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure can be imparted before or after assembly of the polynucleotide.
  • the sequence of nucleotides can be interrupted by non-nucleotide components.
  • polynucleotide can be further modified after polymerization, such as by conjugation with a labeling component.
  • the term also refers to both double- and single-stranded molecules. Unless otherwise specified or required, a polynucleotide encompasses both the double- stranded form and each of two complementary single-stranded forms known or predicted to make up the double-stranded form.
  • a polynucleotide is composed of a specific sequence of four nucleotide bases: adenine (A); cytosine (C); guanine (G); thymine (T); and uracil (U) for thymine when the polynucleotide is RNA.
  • A adenine
  • C cytosine
  • G guanine
  • T thymine
  • U uracil
  • percent identity refers to sequence identity between two peptides or between two nucleic acid molecules. Percent identity can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same base or amino acid, then the molecules are identical at that position.
  • homologous nucleotide sequences include those sequences coding for naturally occurring allelic variants and mutations of the nucleotide sequences set forth herein.
  • homologous nucleotide sequences include nucleotide sequences encoding for a protein of a mammalian species other than humans.
  • Homologous amino acid sequences include those amino acid sequences which contain conservative amino acid substitutions and which polypeptides have the same binding and/or activity. In some embodiments, a homologous amino acid sequence has no more than 15, nor more than 10, nor more than 5 or no more than 3 conservative amino acid substitutions. In some embodiments, a nucleotide or amino acid sequence has at least 60%, at least 65%, at least 70%), at least 80%>, or at least 85%> or greater percent identity to a sequence described herein. In some embodiments, a nucleotide or amino acid sequence has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to a sequence described herein.
  • Percent identity can be determined by, for example, the Gap program (Wisconsin Sequence Analysis Package, Version 8 for UNIX, Genetics Computer Group, University Research Park, Madison Wis.), using default settings, which uses the algorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, 482-489). Algorithms suitable for determining percent sequence identity include the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (Nuc. Acids Res. 25:3389-402, 1977), and Altschul et al. (J. Mol. Biol. 215:403-10, 1990), respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (see the internet at ncbi.nlm.nih.gov).
  • the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.
  • expression refers to the production of a gene product.
  • transient when referred to expression means a polynucleotide is not incorporated into the genome of the cell.
  • cytokine refers to the general class of biological molecules which effect cells of the immune system.
  • cytokines include, but are not limited to, interferons and interleukins (IL), in particular IL-2, IL-12, IL-15, IL-18 and IL-21.
  • IL-2 interferons and interleukins
  • the cytokine is IL-2.
  • vector refers to a non-chromosomal nucleic acid comprising an intact replicon such that the vector may be replicated when placed within a permissive cell, for example by a process of transformation.
  • a vector may replicate in one cell type, such as bacteria, but have limited ability to replicate in another cell, such as mammalian cells.
  • Vectors may be viral or non-viral.
  • Exemplary non-viral vectors for delivering nucleic acid include naked DNA; DNA complexed with cationic lipids, alone or in combination with cationic polymers; anionic and cationic liposomes; DNA-protein complexes and particles comprising DNA condensed with cationic polymers such as heterogeneous polylysine, defined-length oligopeptides, and polyethylene imine, in some cases contained in liposomes; and the use of ternary complexes comprising a virus and polylysine-DNA.
  • the term "antibody” refers to an immunoglobulin or fragment thereof.
  • the antibody may be of any type (e.g., IgG, IgA, IgM, IgE or IgD).
  • the antibody is IgG.
  • An antibody may be non-human (e.g., from mouse, goat, or any other animal), fully human, humanized, or chimeric.
  • antibody fragment refers to any portion of the antibody that recognizes an epitope. Antibody fragments may be glycosylated.
  • the antibody fragment may be a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a Fv fragment, an rlgG fragment, a functional antibody fragment, single chain recombinant forms of the foregoing, and the like.
  • F(ab')2, Fab, Fab' and Fv are antigen- binding fragments that can be generated from the variable region of IgG and IgM. They vary in size, valency, and Fc content.
  • the fragments may be generated by any method, including expression of the constituents (e.g., heavy and light chain portions) by a cell or cell line, or multiple cells or cell lines.
  • the antibody fragment recognizes the epitope and contains a sufficient portion of an Fc region such that it is capable of binding an Fc receptor.
  • cancer refers to all types of cancer, neoplasm, or malignant tumors found in mammals, including leukemia, carcinomas and sarcomas.
  • Exemplary cancers include cancer of the brain, breast, cervix, colon, head & neck, liver, kidney, lung, non-small cell lung, melanoma, mesothelioma, ovary, sarcoma, stomach, uterus and Medulloblastoma.
  • Additional examples include, Hodgkin's Disease, Non-Hodgkin's Lymphoma, multiple myeloma, neuroblastoma, ovarian cancer, rhabdomyosarcoma, primary thrombocytosis, primary macroglobulinemia, primary brain tumors, cancer, malignant pancreatic insulanoma, malignant carcinoid, urinary bladder cancer, premalignant skin lesions, testicular cancer, lymphomas, thyroid cancer, neuroblastoma, esophageal cancer, genitourinary tract cancer, malignant hypercalcemia, endometrial cancer, adrenal cortical cancer, neoplasms of the endocrine and exocrine pancreas, and prostate cancer.
  • anti-tumor vaccine refers to the induction and maintenance of an immune response to a tumor preventing tumor regrowth and/or generation of secondary tumors.
  • NK-92 cells Due to concerns that NK-92 cells might proliferate in the body and cause unwanted side effects, these cells can be irradiated prior to administration to the patient. Irradiated NK-92 cells survive only about 24 to 48 hours after administration to the patient. As the NK-92 cells are likely to target the primary tumor and have limited half-lives, metastatic cells and cancer stem cells may elude this treatment. However, as described herein, administration of NK-92 cells induces an immune response in the subject that is able to produce an anti -tumor vaccine, and that such response persists in the subject after the NK-92 cells have died.
  • NK-92 cells induces an immune response in a subject that is capable of rejecting a tumor upon tumor re-challenge.
  • administration of NK-92 cells at or near the site of a tumor specifically, CAR expressing NK-92 cells, acts as a vaccine against the tumor.
  • CAR-expressing-NK-92 are capable of preventing tumor regrowth.
  • the CAR-expressing NK-92 cells when administered, are sufficient to treat a primary tumor while also eliciting an immune response that prevents potential secondary tumors and/or tumor regrowth.
  • an effective amount of NK-92 cells results in lysis of at least a portion of tumor cells in the primary tumor, and also causes the patient's immune system to recognize antigens from the tumor such that tumor cells are recognized and attacked (e.g., by T cells) even after the NK-92 cells are no longer active in the patient.
  • the therapeutically effective amount of the CAR-expressing NK-92 cells will vary depending on the tumor being treated and its severity as well as the age, weight, etc., of the patient to be treated. The skilled artisan will be able to determine appropriate dosages depending on these and other factors.
  • the compositions can also be administered in combination with one or more additional therapeutic compounds.
  • NK-92 cells to treat a primary tumor in a first location can lead to a prolonged anti-tumor immune response prevents secondary tumors (metastases) at other locations in the patient's body, even after the NK-92 cells have ceased to function.
  • a method of treating cancer in a subject comprising administering to the subject an effective amount of NK-92 cells to induce an immune response in the subject, the immune response is capable of inhibiting generation of secondary tumors.
  • NK-92 cells do not express interleukin 6 (IL-6).
  • IL-6 is a marker of increased immune response in a patient.
  • IL-6 expression is increased in the patient after administration of NK-92 cells.
  • IL-6 expression persists after the administered NK-92 cells cease to function.
  • the tumor may be, for example, a colorectal tumor, a breast tumor, a lung tumor, a prostate tumor, a pancreatic tumor, a bladder tumor, a cervical tumor,
  • cholangiocarcinoma gastric sarcoma, glioma, leukemia, lymphoma, melanoma, multiple myeloma, osteosarcoma, an ovarian tumor, a stomach tumor, a brain tumor.
  • one or more additional cancer treatments or therapies are administered to the subject to treat the primary tumor.
  • a cancer drug is administered to the subject.
  • radiation is administered to the subject.
  • additional cancer treatments are administered, they may be administered prior to,
  • the NK-92 cell line is a unique cell line that was discovered to proliferate in the presence of interleukin 2 (IL-2). Gong et al., Leukemia 8:652-658 (1994). These cells have high cytolytic activity against a variety of cancers.
  • IL-2 interleukin 2
  • NK-92 was discovered in the blood of a subject suffering from a non-Hodgkins lymphoma and then immortalized ex vivo. NK-92 cells are derived from NK cells, but lack the major inhibitory receptors that are displayed by normal NK cells, while retaining the majority of the activating receptors. NK-92 cells do not, however, attack normal cells nor do they elicit an unacceptable immune rejection response in humans. Characterization of the NK-92 cell line is disclosed in WO 1998/49268 and U.S. Patent Application Publication No. 2002-0068044.
  • NK-92 cells can be further engineered to express a chimeric antigen receptor (CAR) on the cell surface.
  • CAR chimeric antigen receptor
  • the CAR is specific for a tumor- specific antigen.
  • Tumor-specific antigens are described, by way of non-limiting example, in US 2013/0189268; WO 1999024566 Al; US 7098008 ; and WO 2000020460 Al, each of which is incorporated herein by reference in its entirety.
  • Tumor-specific antigens include, without limitation, CD 19, CD20, NKG2D ligands, CS1, GD2, CD 138, EpCAM, HER-2, EBNA3C, GPA7, CD244, CA-125, MUC-1, ETA, MAGE, CEA, CD52, CD30, MUC5AC, c-Met, EGFR, FAB, WT-1, PSMA, NY-ESOl, CSPG-4, IGF1-R, Flt-3, CD276, BCMA, CD33, or 41BB.
  • the CAR is a CD19 CAR.
  • polynucleotide and polypeptide sequences for the CD19 CAR are provided in SEQ ID NO:2 and SEQ ID NO:4 (CD19 CAR polynucleotide), and SEQ ID NO:3 and SEQ ID NO:5 (CD19 CAR polypeptide).
  • the CAR comprises an ScFv antigen-binding domain.
  • the CAR comprises an antigen binding domain (e.g., ScFv) that specifically binds an antigen expressed by tumor cells.
  • the antigen binding domain or ScFv specifically binds the following antigens: CD 19, CD20, NKG2D ligands, CS1, GD2, CD138, EpCAM, HER-2, EBNA3C, GPA7, CD244, CA-125, MUC-1, ETA, MAGE, CEA, CD52, CD30, MUC5AC, c-Met, EGFR, FAB, WT-1, PSMA, NY- ESOl, CSPG-4, IGF1-R, Flt-3, CD276, BCMA, CD33, or 41BB.
  • the antigen binding domain or ScFv specifically binds the CD 19 antigen.
  • the CAR comprises an antigen binding domain or ScFv having the following sequences (or a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the following sequences): CD19 (SEQ ID NO:3, SEQ ID NO:5), CD 20 (SEQ ID NO:7); CD33 (SEQ ID NO:9), CSPG4 (SEQ ID NO: 11), EGFR (SEQ ID NO: 13), IGF1R (SEQ ID NO:
  • CD30 SEQ ID NO: 17
  • HER2/neu SEQ ID NO: 19
  • GD2 SEQ ID NO:22 (VL/VH format) or SEQ ID NO:23 (VH/VL format)
  • the CAR comprises a hinge region from CD8.
  • the hinge region comprises the amino acid sequence of SEQ ID NO: 26, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 26.
  • the CAR comprises a transmembrane domain from CD3zeta.
  • the transmembrane domain comprises SEQ ID NO: 28, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 28.
  • the NK-92 cell or cell line is genetically modified with a nucleic acid construct that encodes a CAR described herein.
  • the nucleic acid construct further comprises a promoter that promotes transcription of the nucleic acid sequences.
  • the promoter is an inducible promoter.
  • the nucleic acid construct comprises a nucleic acid sequence that encodes an antigen binding protein (ABP).
  • the ABP is an scFv or a codon optimized scFv.
  • the ABP specifically binds an antigen expressed by a tumor cell.
  • the ABP comprises a region of a CAR described herein (in other words, the CAR comprises the ABP).
  • the construct comprises a nuclei acid that encodes a cytokine, such a IL-2.
  • the cytokine is targeted to the endoplasmic reticulum.
  • the CAR is transiently expressed by the NK-92 cell. In one embodiment, the CAR is stably expressed by the NK-92 cell.
  • K-92 cells are modified to express an Fc receptor protein on the cell surface.
  • Fc receptors include CD64, CD32, CD 16 (e.g., CD 16a and CD 16b), FcsRI, CD23, CD89, Fca/ ⁇ , and FcRn. In some embodiments, the Fc receptor is CD 16.
  • the Fc receptor is a high-affinity Fc receptor comprising a valine at position 158 of the mature protein, or a valine at the position corresponding to position 158 of the mature protein (CD16 F158V).
  • an antibody specific for the target tumor cell is coadministered with the NK-92 cells. Co-administration encompasses administration of the antibody immediately prior to, concurrently with, or immediately after administration of the NK-92 cells.
  • NK-92 cells are modified to express at least one cytokine.
  • the at least one cytokine is IL-2, IL-12, IL-15, IL-18, IL-21, or a variant thereof.
  • the cytokine is IL-12 or a variant thereof.
  • the cytokine is IL-2 or a variant thereof.
  • the cytokine is a variant that is targeted to the endoplasmic reticulum.
  • NK-92 cells can be administered to an individual by absolute numbers of cells, e.g., said individual can be administered from about 1000 cells/injection to up to about 10 billion cells/injection, such as at about, at least about, or at most about, 1 ⁇ 10 8 , 1 ⁇ 10 7 , 5 ⁇ 10 7 , lxlO 6 , 5xl0 6 , lxlO 5 , 5xl0 5 , ⁇ ⁇ ⁇ 4 , 5 ⁇ 10 4 , ⁇ ⁇ ⁇ 3 , 5 ⁇ 10 3 (and so forth) NK-92 cells per injection, or any ranges between any two of the numbers, end points inclusive.
  • NK-92 cells can be administered to such an individual by relative numbers of cells, e.g., said individual can be administered about 1000 cells to up to about 10 billion cells per kilogram of the individual, such as at about, at least about, or at most about, 1 ⁇ 10 8 , lxlO 7 , 5xl0 7 , lxlO 6 , 5xl0 6 , ⁇ ⁇ ⁇ 5 , 5 ⁇ 10 5 , ⁇ ⁇ ⁇ 4 , 5 ⁇ 10 4 , ⁇ ⁇ ⁇ 3 , 5 ⁇ 10 3 (and so forth) NK-92 cells per kilogram of the individual, or any ranges between any two of the numbers, end points inclusive.
  • the total dose may calculated by m 2 of body surface area, including lxlO 11 , lxlO 10 , lxlO 9 , lxlO 8 , lxlO 7 , perm 2 .
  • the average person is 1.6-1.8 m .
  • the methods include treating a primary tumor while inducing and maintaining an immune response to the tumor in the subject.
  • the methods include administering to the subject an effective amount of CAR-expressing-NK-92 cells to treat the primary tumor thereby inducing an anti-tumor immune response that is maintained in the subject.
  • the maintained immune response prevents tumor regrowth and/or inhibits generation of secondary tumors.
  • interleukin 6 expression is increased in the patient.
  • the CAR-expressing-NK-92 cells induce lysis of tumor cells in the primary tumor.
  • a cytokine is co-administered to the subject.
  • the cytokine is interleukin 2.
  • the cytokine is interleukin 12.
  • a chemotherapeutic agent is administered to the subject prior to administration of the CAR- expressing-NK-92 cells.
  • the CAR-expressing-NK-92 cells are administered systemically.
  • the CAR-expressing-NK-92 cells are administered proximate to or directly into the primary tumor.
  • the tumor is selected from the group consisting of colorectal tumor, breast tumor, lung tumor, prostate tumor, pancreatic tumor, bladder tumor, cervical tumor, cholangiocarcinoma, gastric sarcoma, glioma, leukemia, lymphoma, melanoma, multiple myeloma, osteosarcoma, ovarian tumor, stomach tumor, brain tumor.
  • the method further includes administering to the subject a cancer drug or radiation to the patient.
  • the subject is selected from the group consisting of bovines, swine, rabbits, alpacas, horses, canines, felines, ferrets, rats, mice, fowl and buffalo.
  • the subject is human.
  • the CAR-expressing-NK-92 cells are mCD19CAR-expressing NK-92 cells.
  • the tumor is a B-cell lymphoma.
  • Also provided are methods of producing an anti -tumor vaccine in a subject with a tumor comprising administering to the subject an effective amount of CAR- expressing-NK-92 cells to the subject thereby inducing an anti-tumor vaccine to the tumor in the subject.
  • interleukin-6 expression is increased in the subject
  • the CAR-expressing-NK-92 cells treats the tumor in the subject.
  • a cytokine is coadministered to the subject.
  • the cytokine is interleukin 2.
  • the cytokine is interleukin 12.
  • a chemotherapeutic agent is administered to the subject prior to administration of the CAR-expressing- K-92 cells.
  • the CAR- expressing- K-92 cells are administered systemically.
  • the CAR-expressing-NK- 92 cells are administered proximate to or directly into the tumor.
  • the tumor is selected from the group consisting of colorectal tumor, breast tumor, lung tumor, prostate tumor, pancreatic tumor, bladder tumor, cervical tumor, cholangiocarcinoma, gastric sarcoma, glioma, leukemia, lymphoma, melanoma, multiple myeloma, osteosarcoma, ovarian tumor, stomach tumor, brain tumor.
  • the method further includes administering to the subject a cancer drug or radiation.
  • the CAR-expressing-NK-92 cells are mCD19CAR-expressing NK-92 cells.
  • the tumor is a B-cell lymphoma.
  • the CAR-expressing NK-92 cells are administered systemically to the subject, e.g. by intravenous injection.
  • the CAR-expressing NK-92 cells are administered locally to the site of a tumor, e.g., intraperitoneal administration or injection of NK-92 cells proximate to or directly into the tumor.
  • Benefits of local administration of CAR- expressing NK-92 cells include, but are not limited to, the ability to use fewer cells to obtain an effect and an increased concentration of CAR-expressing NK-92 cells at the tumor site.
  • a cytokine or multiple cytokines are administered to the subject concurrently with the CAR-expressing NK-92 cells.
  • the cytokine is a cytokine that further stimulates an immune response.
  • the cytokine is a cytokine that elicits a T cell and/or NK cell response.
  • the cytokine is IL-2 and/or IL-12. Without being bound by theory, it is believed that administration of cytokines will further elicit a T cell response against the tumor and/or potentiate CAR-expressing NK-92 cell activity.
  • the cytokine is administered systemically to the patient.
  • the cytokine is administered locally to the site of the primary tumor.
  • the cancer is selected from the group consisting of leukemia
  • acute leukemias e.g., acute lymphocytic leukemia, acute myelocytic leukemia (including myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemia
  • chronic leukemias e.g., chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia
  • polycythemia vera e.g., lymphomas (e.g., Hodgkin's disease and non- Hodgkin's disease), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, solid tumors including, but not limited to, sarcomas and carcinomas such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphan
  • K-92 cells can be administered to an individual by absolute numbers of cells, e.g., said individual can be administered from about 1000 cells/injection to up to about 10 billion cells/injection, such as at about, at least about, or at most about, 1 ⁇ 10 8 , 1 ⁇ 10 7 , 5 ⁇ 10 7 , l x lO 6 , 5x l0 6 , l x lO 5 , 5x l0 5 , ⁇ ⁇ ⁇ 4 , 5 ⁇ 10 4 , ⁇ ⁇ ⁇ 3 , 5 ⁇ 10 3 (and so forth) NK-92 cells per injection, or any ranges between any two of the numbers, end points inclusive.
  • said individual can be administered from about 1000
  • cells/injection/m 2 to up to about 10 billion cells/injection/m 2 , such as at about, at least about, or at most about, 1 ⁇ 10 8 /m 2 , 1 x 10 7 /m 2 , 5 x 10 7 /m 2 , 1 x 10 6 /m 2 , 5 x 10 6 /m 2 , 1 x 10 5 /m 2 , 5 x 10 5 /m 2 , 1 x lOVm 2 , 5x 10 4 /m 2 , 1 10 3 /m 2 , 5x 10 3 /m 2 (and so forth) NK-92 cells per injection, or any ranges between any two of the numbers, end points inclusive.
  • NK-92 cells can be administered to such individual by relative numbers of cells, e.g., said individual can be administered about 1000 cells to up to about 10 billion cells per kilogram of the individual, such as at about, at least about, or at most about, l x lO 8 , l x lO 7 , 5x l0 7 , l x lO 6 , 5 ⁇ 10 6 , ⁇ ⁇ ⁇ 5 , 5 ⁇ 10 5 , ⁇ ⁇ ⁇ 4 , 5 ⁇ 10 4 , ⁇ ⁇ ⁇ 3 , 5 ⁇ 10 3 (and so forth) NK-92 cells per kilogram of the individual, or any ranges between any two of the numbers, end points inclusive.
  • the total dose may calculated by m 2 of body surface area, including about 1 x 10 11 , 1 x 10 10 , 1 x 10 9 , 1 x 10 8 , 1 x 10 7 , per m 2 , or any ranges between any two of the numbers, end points inclusive.
  • the average person is about 1.6 to about 1.8 m 2 .
  • between about 1 billion and about 3 billion NK-92 cells are administered to a patient.
  • the amount of NK-92 cells injected per dose may calculated by m 2 of body surface area, including 1 10 11 , 1 ⁇ 10 10 , 1 ⁇ 10 9 , 1 ⁇ 10 8 , 1 ⁇ 10 7 , per m 2 .
  • the average person is 1.6-1.8 m 2 .
  • NK-92 cells The NK-92 cells, and optionally other anti-cancer drugs (e.g., IL-12), and optionally other anti-cancer drugs (e.g.
  • chemotherapeutic agents can be administered once to a patient with cancer can be administered multiple times, e.g., once every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 hours, or once every 1, 2, 3, 4, 5, 6 or 7 days, or once every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more weeks during therapy, or any ranges between any two of the numbers, end points inclusive.
  • NK-92 cells are administered in a composition comprising NK-92 cells and a medium, such as human serum or an equivalent thereof.
  • the medium comprises human serum albumin.
  • the medium comprises human plasma.
  • the medium comprises about 1% to about 15% human serum or human serum equivalent.
  • the medium comprises about 1% to about 10% human serum or human serum equivalent.
  • the medium comprises about 1% to about 5% human serum or human serum equivalent.
  • the medium comprises about 2.5% human serum or human serum equivalent.
  • the serum is human AB serum.
  • a serum substitute that is acceptable for use in human therapeutics is used instead of human serum. Such serum substitutes are known in the art. Although concentrations of human serum over 15%) can be used, it is contemplated that concentrations greater than about 5%> will be cost-prohibitive.
  • NK-92 cells including modified NK-92 cells are administered in a composition comprising NK-92 cells and an isotonic liquid solution that supports cell viability.
  • NK-92 cells are administered in a composition that has been reconstituted from a cryopreserved sample.
  • Pharmaceutically acceptable compositions can include a variety of carriers and excipients.
  • a variety of aqueous carriers can be used, e.g., buffered saline and the like. These solutions are sterile and generally free of undesirable matter. Suitable carriers and excipients and their formulations are described in Remington: The Science and Practice of Pharmacy, 21st Edition, David B.
  • pharmaceutically acceptable carrier is meant a material that is not biologically or otherwise undesirable, i.e., the material is administered to a subject without causing undesirable biological effects or interacting in a deleterious manner with the other components of the pharmaceutical composition in which it is contained. If administered to a subject, the carrier is optionally selected to minimize degradation of the active ingredient and to minimize adverse side effects in the subject.
  • pharmaceutically acceptable is used synonymously with physiologically acceptable and pharmacologically acceptable.
  • a pharmaceutical composition will generally comprise agents for buffering and preservation in storage and can include buffers and carriers for appropriate delivery, depending on the route of
  • compositions for use in in vivo or in vitro may be sterilized by conventional, well-known sterilization techniques.
  • the compositions may contain acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents and the like, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like.
  • concentration of cells in these formulations and/or other agents can vary and will be selected primarily based on fluid volumes, viscosities, body weight and the like in accordance with the particular mode of administration selected and the subject's needs.
  • the NK-92 cells are administered to the patient in conjunction with one or more other treatments for the cancer being treated.
  • one or more other treatments for the cancer being treated includes, for example, an antibody, radiation, chemotherapeutic, stem cell transplantation, or hormone therapy.
  • an antibody is administered to the patient in conjunction with the K-92 cells.
  • the K-92 cells and an antibody are administered to the patient together, e.g., in the same formulation; separately, e.g., in separate formulations, concurrently; or can be administered separately, e.g., on different dosing schedules or at different times of the day.
  • the antibody can be administered in any suitable route, such as intravenous or oral administration.
  • additional therapeutic agents can be used that are suitable to the disease being treated.
  • the provided methods of treatment further comprise administering a second therapeutic agent to the subject.
  • additional therapeutic agents include, but are not limited to, analgesics, anesthetics, analeptics, corticosteroids, anticholinergic agents, anticholinesterases, anticonvulsants, antineoplastic agents, allosteric inhibitors, anabolic steroids, antirheumatic agents, psychotherapeutic agents, neural blocking agents, anti-inflammatory agents, antihelmintics, antibiotics, anticoagulants, antifungals, antihistamines, antimuscarinic agents, antimycobacterial agents, antiprotozoal agents, antiviral agents, dopaminergics,
  • agent and dosage can be determined readily by one of skill in the art based on the given disease being treated.
  • Combinations of agents or compositions can be administered either concomitantly (e.g., as a mixture), separately but simultaneously (e.g., via separate intravenous lines) or sequentially (e.g., one agent is administered first followed by administration of the second agent).
  • concomitantly e.g., as a mixture
  • sequentially e.g., one agent is administered first followed by administration of the second agent.
  • combination is used to refer to concomitant, simultaneous or sequential administration of two or more agents or
  • compositions are preferred.
  • the course of treatment is best determined on an individual basis depending on the particular characteristics of the subject and the type of treatment selected.
  • the treatment such as those disclosed herein, can be administered to the subject on a daily, twice daily, bi-weekly, monthly or any applicable basis that is therapeutically effective.
  • the treatment can be administered alone or in combination with any other treatment disclosed herein or known in the art.
  • the additional treatment can be administered simultaneously with the first treatment, at a different time, or on an entirely different therapeutic schedule (e.g., the first treatment can be daily, while the additional treatment is weekly).
  • any subset or combination of these is also specifically contemplated and disclosed. This concept applies to all aspects of this disclosure including, but not limited to, steps in methods using the disclosed compositions. Thus, if there are a variety of additional steps that can be performed, it is understood that each of these additional steps can be performed with any specific method steps or combination of method steps of the disclosed methods, and that each such combination or subset of combinations is specifically
  • Example 1 NK-92 cytokine production in vitro
  • NK-92 production of a variety of cytokines was determined by qualitative ELISA assay. Results are shown in Fig. 1. NK-92 cells produce IL-8, IL-10, and interferon gamma (IFNy). NK-92 cells do not produce assayable amounts of IL-6.
  • NK-92 cells were transduced with a retrovirus coding for a second generation anti-murine CD19-CAR. Significant amounts of CD19-CAR were detectable by flow cytometry in whole NK-92 cells (Fig. 2A).
  • mCD19CAR-NK-92 cells were added to A20 murine lymphoma cells at effectontarget (E:T) ratios ranging from 0.15 : 1 to 20: 1.
  • E:T effectontarget
  • mCD19CAR-NK-92 cells killed a greater percentage of A20 lymphoma cells as compared to wild type NK-92 cells (Fig. 2B).
  • E:T ratios resulted in increased killing of A20 lymphoma cells.
  • Example 3 In vivo development of immune response after localized administration of NK-92 cells in mice
  • A20 murine lymphoma cells (A20 are murine CD19 + ). After tumor was established (at approximately days 6 to 8), mice were injected intra-tumor either with a control or NK-92 cells (Fig. 3 : PBS control, triangle; wild type, star; CD19CAR, circle) at days 13 and 15 post- lymphoma cell injection. The mouse administered CD19CAR was re-challenged twice with A20 murine lymphoma cells on the opposite flank from the original injection at
  • Results are shown in Fig. 3.
  • Tumor surface area (mm 2 ) was reduced after the first NK-92-CD19CAR administration and continued regression until the tumor was no longer visible.
  • Tumor surface area was reduced after the second injection of wild type NK-92 cells, but not the first, and the tumor resumed growth within several days of the second NK- 92 cell injection.
  • lymphoma re-challenge in the animal administered NK-92- CD19CAR did not result in tumor growth, regardless of the site of lymphoma cell injection, even though the NK-92-CD19CAR cells would be expected to have ceased activity in the mouse at the time of re-challenge.
  • Example 4 Antitumor Vaccination Using CD19-CAR-Expressing NK-92 Cells in Treatment of Subcutaneous A20 B-Cell Lymphoma in Balb/c Mice.
  • NK-92 cells can be successfully redirected to specifically kill target cells from murine origin through expression of an anti- murine CAR.
  • mCD19CAR induces clearance of s.c. A20 lymphoma cell tumors and significantly improves survival.
  • Successful tumor clearance correlates with resistance to later challenges with A20 cells, indicating the development of a long-term immune response in the treated mice.
  • Resistance to A20 cells re-challenges appears to be independent on T-cells.
  • NK-92 cells were transduced with a lentivirus construct coding for a third generation anti -murine CD 19 CAR and injected intra- tumorally (5xl0e6 NK cells, two injections three days apart) into a murine syngeneic subcutaneous lymphoma (10e6 A20 cells into BALB/c mice). Tumor size was monitored over time and mice showing clearance of the tumors were re-challenged with another subcutaneous injection of A20 cells contralaterally.
  • Targeted activated NK-92 cells (mCD 19-CAR taNK) effectively killed murine cancer cells in vitro (>60% killing at E:T ratio of 5: 1).
  • intra-tumor injection of mCD19CAR taNK induced significant tumor regression compared to saline (342 mm3 and 936 mm3 respectively at day 16, p ⁇ 0.05) and significantly improved survival, with 75% of the mice showing complete tumor regression (p ⁇ 0.05).
  • injection of parental NK cells did not significantly affect tumor size in mice (815 mm3 at day 16).
  • re- challenge of the tumor-free mice with A20 cells failed to induce tumor regrowth after 14 days in 5 out of 6 mice, suggesting induction of a memory (“vaccine”) effect after the injection of mCD19 ta K.
  • mCD19 ta K can effectively kill CD19-positive murine cancer cells.
  • intra- tumor injections of targeted mCD19CAR taNK into a fully immunocompetent mouse model can induce tumor clearance and protection from tumor re-challenge.
  • Fig. 4A is a schematic showing the experimental design.
  • mice were anesthetized with isoflurane and injected with 2.5 x 10 6 A20 murine lymphoma cells in 100 ⁇ _, volume of serum free media, subcutaneously (s.c.) into the left flank. Beginning two days after tumor cell inoculation, tumors were measured daily by digital caliper. Ten days after inoculation, single inoculation animals of the same sex were randomized around a mean tumor volume ( ⁇ 140mm 3 at time of randomization) into Groups 1-6, with each group consisting of animals bearing a similar mean tumor volume and range.
  • Tumors were measured three times each week (3x/week) by digital caliper to monitor tumor growth, and animals were weighed and monitored daily for general health and survival, and assigned a Body Condition Score (see Body Condition Scoring) once a week (lx/week). Any animal bearing a tumor of volume > 1500mm 3 or a tumor that has ulcerated; or any animal that has lost > 30% of its body weight at Day 0; or displays a Body Condition Score of ⁇ 2; or is moribund were euthanized by CO2 overdose. Changes in tumor volumes for male and female mice treated with vehicle, NK-92, or mCD19CAR-NK-19 are plotted in Fig. 4C. Mouse survival is shown in Fig. 4D.
  • Taconic Biosciences aged 5-6 weeks, with mean body weight ( ⁇ SD) of 21.65g ⁇ 2.47 on Day 0 were used. Animals were uniquely identified using an ear punch. Animals were acclimatized at least 3 days prior to study commencement. During this period, the animals were observed daily in order to reject animals that were in poor condition.
  • Walls and cage racks were sponged a minimum of once per month with a dilute bleach solution.
  • a cage card or label with the appropriate information necessary to identify the study, dose, animal number and treatment group marked all cages. The temperature and relative humidity was recorded during the study, and the records retained.
  • mean tumor volumes (mm 3 + SEM) for enrolled animals from each group were as follows: Group 1 : 149.22 + 50.20; Group 2: 128.02 + 64.94; Group 3 : 158.68 + 95.54; Group 4: 129.73 + 30.60; Group 5: 149.58 + 65.50; Group 6: 128.46 + 48.71.
  • A20 cells were provided by the Sponsor. Cells were counted by hemocytometer/trypan-blue exclusion and 2.5 x 10 6 A20 murine lymphoma cells in ⁇ . volume of serum free media injected subcutaneously (s.c.) into the left flank.
  • Cells were delivered by 25G needle, inserted so that the tip was at the approximate center of the tumor mass.
  • Taconic Biosciences aged 5-6 weeks were enrolled in the study. All animals were housed under standard environmental conditions in groups of five (5) animals each of the same sex, with even numbered Groups consisting of females and odd numbered Groups consisting of males, and maintained on appropriate rodent chow and sterile water ad libitum. Mice were anesthetized with isoflurane and injected with 2.5 x 10 6 A20 murine lymphoma cells in ⁇ . volume of serum free media, subcutaneously (s.c.) into the left flank. Beginning on two days after tumor cell inoculation, tumors were measured daily by digital caliper.
  • Tumors were measured three times each week (3x/week) by digital caliper to monitor tumor growth, and animals were weighed and monitored daily for general health and survival, and assigned a Body Condition Score (see Body Condition Scoring) once a week (lx/week). Any animal bearing a tumor of volume > 1500mm 3 or a tumor that has ulcerated; or any animal that has lost > 30% of its body weight at Day 0; or displays a Body Condition Score of ⁇ 2; or is moribund will be euthanized by CO2 overdose.
  • Tumor Seeding Tumor seeding efficiency was assessed on Day 0, prior to randomization. Any animal not developing a tumor exceeding 50mm 3 by study completion was excluded from the study. Such tumors were considered inviable.
  • Animal Survival Animals were monitored for survival daily. Any animal requiring euthanasia according to animal health and welfare thresholds, including loss of greater than 30% of their initial body weight, tumors exceeding 1500mm 3 or ulcerated, inability to obtain food/water or moribund was included for representation of survival and statistical analysis.
  • Tumor Measurement Tumors were measured three times each week
  • Body Condition Scoring A Body Condition Score was assigned to all animals lx/week, using the following criteria:
  • the animal is emaciated, skeletal structure very prominent with little
  • Vertebrae are distinctly segmented.
  • Fig. 4A shows the experimental design. Tumor seeding was assessed on Day 0, and again at study completion. Any tumor that did not exceed of a volume of 50mm 3 was considered inviable.
  • Fig. 4B shows tumor take rate following subcutaneous injection of 2.5 x 10 6 A20 Balb/c murine lymphoma cells into Balb/c mice was 100% (25/25) for male mice and 92% (23/25) for female mice. This difference was not statistically significant when tested by two-tailed Fisher's exact test. These observations suggest that animal gender does not significantly impact tumor seeding/rejection in this model.
  • Animal Survival Animals were monitored for survival daily through Day
  • mice All animal deaths in the study were euthanizations due to tumors exceeding animal welfare thresholds of 1500mm 3 volumes or ulceration. Groups were considered by gender, with changes in tumor volume shown in Fig. 4C. Survival of mice is shown in Fig. 4D.
  • tumors treated with parental K-92 cells displayed similar steady growth kinetics to tumors treated with vehicle (Group 1), with mean tumor volumes exceeding 1000mm 3 by Day 20.
  • single A20 tumors of animals treated with mCD19-CAR- K-92 cells displayed observably inhibited growth kinetics compared to control, with mean tumor volumes not exceeding 1000mm 3 through the full course of the study.
  • NK-92 cells For the female animals, animals bearing single tumors treated with either parental NK-92 cells (Group 4) or mCD19-CAR-NK-92 cells (Group 6) displayed inhibited growth kinetics compared to control; indeed mean tumor volumes of Group 6 animals did not exceed 1000 mm 3 through the full course of the study.
  • Fig. 5 shows average tumor volumes at Day 16.
  • Tumor Re-challenge To test whether functional immunological memory might persist in animals previously exposed to A20 tumors, animals in Groups 1-6 showing a complete response (i.e. elimination of s.c. tumor mass) were re-challenged with a second s.c. injection of 2.5 x 10 6 A20 cells into the right flank on Day 30.
  • the tumor take rate data from the rechallenge portion of the study are summarized in Table 2.
  • Fig. 6 shows all tumor-free mice were re-challenged by a subcutaneous injection of A20 cells in the contralateral flank. All mice remained tumor-free and survived until day 60 post-treatment, except one.
  • mice in the mCD19CAR-treated arm appeared tumor-free by day 30 post-treatment, compared to 1/10 mice in NK-92 treated arm and 1/9 in vehicle treated arm.
  • Example 5 CD19 CAR NK-92 cells (CD19 taNK) Induce Complete Remissions in a Highly Aggressive Murine Lymphoma Model (L1210) With Effective Protection Against Re-challenge
  • the L1210 malignant lymphoma cell line derived from DBA/2 mice, is notable both for its short doubling time of 8-10 hours and for its long history of successful use by the NCI in the identification of effective clinical treatments for hematological malignancies.
  • aNK clinical grade NK-92
  • mice used in this Example were DBA/2J male mice, 6-8 weeks of age. All mice were injected with tumor cells on Day PRO. 8 days after tumor cell implantation, when mean tumor volumes were measured to be between 50 and 150 mm 3 , thirty (30) animals bearing tumors of -90 mm 3 were selected for enrollment in the study, and these animals were randomized into three (3) groups consisting of ten (10) animals each, with animals in each group bearing tumors of similar mean volume and volume range. Randomization day was considered Day 0 of the study, and treatments were commenced on this day.
  • Animals in Group 1 were administered vehicle (serum free DMEM) as an intratumoral (i.t.) injection of 50 ⁇ 1.
  • Animals in Group 2 were administered 2 x 106 K-92.C cells i.t. in a volume of 50 ⁇ 1.
  • Animals in Group 3 were administered 2 x 106 mCD19-CAR- NK-92 cells i.t. in a volume of 50 ⁇ 1. Identical treatments were administered on Days 0, 2 and 4 of the study. Additional details of intratumoral administration are provided in Methods, below.
  • the vehicle was ⁇ serum-free DMEM.
  • L1210-Luc cells were grown under tissue-culture conditions in DMEM supplemented with 10% Horse Serum. The area around the subcutaneous injection sites was shaved prior to injection of tumor cells. On Day PRO, cells were counted by
  • Animals were injected intratumorally (i.t.) with 2 x 10 6 test cells in 50 ⁇ 1 volume on Days 0, 2 and 4 as indicated in Table 3. Briefly, cells were delivered by 25G needle, inserted so that the tip was at the approximate center of the tumor mass. The cells were slowly released into the tumor, and the needle was held in place for a minimum of 30 seconds following dose to allow for the volume to be absorbed into the tumor. The needle was slowly withdrawn as the tumor was squeezed between forceps at the entry site to prevent leakage of the dose from the entry. The forceps was held in place following needle exit for an additional 30 seconds.
  • Example 6 This example demonstrates that treatment of mice having A20 tumors with mCD19-CAR-NK-92 cells increased survival, and mice that completely responded to treatment rejected A20 tumor allografts when re-challenged.
  • mice Forty (40) 5-7 week old BALB/c mice (20 males and 20 females) were sourced Taconic Biosciences to serve Part A.
  • PR pre-randomization
  • animals were injected subcutaneously (s.c.) into the left flank with 2.5 x 10 6 A20 murine lymphoma cells in 100 ⁇ _, volume of serum free media. Beginning on Day PR7, tumors were measured daily.
  • mice were injected intratumorally (i.t.) with test cells or vehicle in 50 ⁇ volume of serum free media into the tumor mass of each animal according to pre-established i.t. procedure (see Experimental Procedures). Briefly, animals were administered vehicle only or were administered 5 x 106 mCD19-CAR- K-92.
  • Part B began on Day 26. Animals from Part A without tumors were enrolled in Part B, along with twelve (12) naive animals (6 males and 6 females). All Part B animals were administered 2.5 x 10 6 A20 cells into the right flank. Tumors were measured 2 times/week. Animals were euthanized on Day 57.
  • Example 7 This example demonstrates that treatment of mice having L1210 tumors with CD19-CAR-NK-92 cells increased survival, and mice that completely responded to treatment rejected L1210 tumor allografts when re-challenged.
  • Animal Survival to Welfare Thresholds - Initial Tumor Challenge [0193] Animals were monitored for survival daily. Animals requiring euthanasia according to animal health and welfare thresholds, including loss of greater than 30% of their initial body weight, tumors exceeding 2500 mm 3 , inability to obtain food/water, or found moribund, were included for survival analysis. Animals requiring euthanasia due to ulcerated tumors were not included in survival analysis.
  • L1210 is an extremely fast-growing, aggressive tumor cell line and 0% of vehicle treated control animals survived further than twenty-three (23) days post tumor challenge.
  • treatment with CD19-CAR-a K cells enhanced survival compared to treatment with vehicle. Indeed, 25% (2/8) of animals treated with CD19-CAR-a K cells survived through study completion at Day 61 through tumor graft challenge.
  • Genbank accession numbers described herein are incorporated by reference herein.
  • SEQ ID NO: 24 High Affinity Variant Immunoglobulin Gamma Fc Region Receptor lll-A amino acid sequence (full length form).
  • SEQ ID NO: 25 High Affinity Variant Immunoglobulin Gamma Fc Region Receptor lll-A nucleic acid sequence (full length form).
  • SEQ ID NO: 28 Human T-cell surface glycoprotein CD3 zeta chain isoform 2 precursor

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Abstract

L'invention concerne des méthodes destinées à induire et à maintenir une réponse immunitaire à une tumeur chez un sujet tout en traitant une tumeur primaire. Les procédés consistent à administrer au sujet une quantité efficace de cellules NK-92 exprimant CAR pour traiter la tumeur primaire, ce qui permet d'induire une réponse immunitaire antitumorale qui est maintenue chez le sujet, la réponse immunitaire maintenue empêchant la reprise de la croissance de la tumeur et/ou inhibant la formation de tumeurs secondaires. L'invention concerne également des procédés de production d'un vaccin antitumoral chez un sujet atteint d'une tumeur. Les procédés consistent à administrer au sujet une quantité efficace de cellules NK-92 exprimant CAR, ce qui permet d'induire un vaccin antitumoral à la tumeur chez le sujet.
EP18815377.9A 2017-11-01 2018-10-31 Cellules nk-92 pour stimuler un vaccin anticancéreux Pending EP3703735A1 (fr)

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